Small molecules that react chemoselectively with a unique non-enzyme protein in a complex biological environment are rare. Such molecules are envisioned to have imaging, pharmacology and protein engineering applications. Pharmacologic examples include Plavix® (clopidogrel) and related thienopyridines, which are oxidized in the liver yielding a thiol that forms a disulfide with a Cys residue in the P2Y12 receptor, inactivating it. [Savi et al., Thromb. Haemost. 84:891-896 (2000)]. There are β-amino ketones identified by high throughput screening that β-eliminate to form α,β-unsaturated ketones and then react with a proximal Cys residue in the thyroid hormone receptor, inhibiting it. [Estebanez-Perpina et al., Mol. Endocrinol. 21:2919-2928 (2007)] There are also small molecules that are known to bind to and react with one Lys ε-amino group within a purified antibody in buffer. [Guo et al., Proc. Natl. Acad. Sci. U.S.A. 103:11009-11014 (2006)] These conjugates, after purification, are envisioned to become injectable drugs (see, covx.com). However, compounds that react with a specific Lys ε-amino group in a non-enzyme protein in the context of a complex biological sample have not been reported.
For pharmacologic applications, it would be advantageous if an orally bioavailable, appropriately reactive small molecule formed a conjugate with an endogenous non-enzyme protein. This strategy can be used to conjugate potent drug candidate substructures, whose individual pharmacologic properties are undesirable, to an endogenous protein such that the conjugate exhibited activity and a favorable half-life and distribution. Lys ε-amine covalent modifiers can also be used to modulate non-enzyme function. A third application is focused upon in this invention, the creation of Lys ε-amine covalent modifiers that form a conjugate with an endogenous protein inhibiting it from aggregating and leading to a gain-of-toxic-function amyloid disease.
Human amyloid diseases, like the transthyretin (TTR) amyloidoses, are named after the characteristic extracellular cross-β-sheet amyloid fibril deposits that result from the mis-assembly of a specific protein. [Cohen et al., Science 313:1604-1610 (2006); and Hardy et al., Science 297:353-356 (2002)] The amyloidogenesis-associated cytotoxicity that appears to be central to amyloid disease etiology is linked to an aging-associated decline in cellular protein homeostasis, or proteostasis, capacity. [Cohen et al., Science 313:1604-1610 (2006); and Balch et al., Science 319:916-919 (2008)].
Senile systemic amyloidosis (SSA) affecting >10% of the aged population, results from wild type (WT) TTR amyloidogenesis leading to cardiomyopathy. [Johnson et al., Acc. Chem. Res. 38:911-921 (2005); and Westermark et al., Proc. Natl. Acad. Sci. U.S.A. 87:2843-2845 (1990)] Deposition of a destabilized TTR mutant causes earlier onset familial amyloid polyneuropathy (FAP) and/or cardiomyopathy (FAC). [Coelho, Curr. Opin. Neurol. 9:355-359 (1996); Jacobson et al., N. Engl. J. Med. 336:466-473 (1997); and Sekijima et al., Cell 121:73-85 (2005)]. No effective treatment is available for SSA or FAC.
The only strategy to ameliorate FAP is gene therapy mediated by liver transplantation, wherein the wild type/mutant TTR (WT-TTR/mutant-TTR) genes in the liver (that synthesize plasma-bound TTR) are replaced by WT-TTR/WT-TTR genes [Holmgren et al., Lancet 341:1113-1116 (1993)], resulting in a dramatic reduction in amyloidogenic TTR concentration in the blood. However, continued WT-TTR deposition in the heart post-transplantation results in cardiomyopathy, limiting effectiveness. Thus, general chemotherapeutic approaches for the TTR amyloidoses are sought. [Johnson et al., Acc. Chem. Res. 38:911-921 (2005)].
Transthyretin is a 55 kDa homotetrameric protein composed of 127-residue β-sheet-rich subunits. [Klabunde et al., Nat. Struct. Biol. 7:312-321 (2000)]. TTR transports thyroxine (T4) and holoretinol binding protein (REP) in the blood and in the cerebrospinal fluid (CSF), using non-overlapping binding sites. [Monaco et al., Science 268:1039-1041 (1995); and Wojtczak et al., J. Biol. Chem. 267:353-357 (1992)] Transthyretin is also present within neurons. Concentrations of TTR in healthy adults have been reported to be in the range of 0.017-0.025 g/L in the CSF [Davidsson et al., J. Neural. Transm. 1997, 104:711-720; Riisoen; Acta Neurol. Scand. 1988, 78:455-459; and Ingenbleek et al., Annu. Rev. Nutr. 1994, 14:495-533] and 0.20-0.40 g/L in the plasma [Hamilton et al., Cell Mol Life Sci 2001, 58:1491-1521].
The more labile dimer-dimer interface of the TTR tetramer creates two funnel-shaped T4 binding sites. Although TTR transports T4 and holoretinol binding protein in the blood and cerebrospinal fluid of mammals, >99% of the T4 sites in human blood are unoccupied because the vast majority of T4 in the blood is carried by thyroid binding globulin and albumin, the latter being most abundant plasma protein. [Klabunde et al., Nat. Struct. Biol. 2000, 7:312; Johnson et al., Acc. Chem. Res. 2005, 38:911; Monaco et al., Science 1995, 268:1039; Wojtczak et al., J. Biol. Chem. 1992, 267:353; and Bartalena et al., Clin. Lab. Med. 1993, 13: 583]. TTR tetramer dissociation is rate limiting for the generation of partially folded monomers that spontaneously self-assemble into TTR amyloid fibrils. [Johnson et al., Acc. Chem. Res. 38:911-921 (2005); Colon et al., Biochemistry 31:8654-8660 (1992); Liu et al., Nat. Struct. Biol. 7:754-757 (2000); Jiang et al., Biochemistry 40:11442-11452 (2001); Hammarstrom et al., Science 299:713-716 (2003); Hammarstrom et al., Science 293:2459-2462 (2001); Hurshman et al., Biochemistry 43:7365-7381 (2004); and Babbes et al., Biochemistry 47:6969-6984 (2008)]. Small molecules that bind to tetrameric TTR and stabilize the ground state more than the dissociative transition state inhibit amyloidogenesis by making the tetramer dissociation barrier insurmountable. [Klabunde et al., Nat. Struct. Biol. 2000, 7:312; Petrassi et al., J. Am. Chem. Soc. 2000, 122:2178; Razavi et al., Angew. Chem. Int. Ed. Engl. 2003, 42:2758; Purkey et al., Chem. Biol. 2004, 11:1719; Johnson et al., Acc. Chem. Res. 2005, 38:911; Johnson et al., J. Med. Chem. 2008, 51:260; Johnson et al., J. Med. Chem. 2008, 51:6348; Adamski-Werner et al., J. Med. Chem. 2004, 47:355; Oza et al., J. Med. Chem. 2002, 45:321; Baures et al., Bioorg. Med. Chem. 1999, 7:1339; Green et al., J. Am. Chem. Soc. 2003, 125:13404; and Hammarstrom et al., Science 2003, 299:713]. These so-called TTR kinetic stabilizers have recently been demonstrated to be efficacious in a phase II/III placebo-controlled clinical trial for familial amyloid polyneuropathy, one of the transthyretin amyloid diseases (See, foldrx.com).
Reversible occupancy of one of the two T4 binding sites by a high affinity small molecule is known to be sufficient to kinetically stabilize the entire TTR tetramer through differential stabilization of the ground state over the dissociative transition state, thus inhibiting amyloidogenesis. [Johnson et al., Acc. Chem. Res. 38:911-921 (2005); Hammarstrom et al., Science 299:713-716 (2003); Hammarstrom et al., Science 293:2459-2462 (2001); Adamski-Werner et al., J. Med. Chem. 47:355-374 (2004); Johnson et al., J. Med. Chem. 51:6348-6358 (2008); Johnson et al., J. Med. Chem. 48:1576-1587 (2005); Miroy et al., Proc. Natl. Acad. Sci. U.S.A. 93:15051-15056 (1996); Oza et al., J. Med. Chem. 45, 321-332 (2002); Razavi et al., Angew. Chem. Int. Ed. Engl. 42, 2758-2761 (2003); Wiseman et al., J. Am. Chem. Soc. 127:5540-5551 (2005); Foss et al., J. Mol. Biol. 347, 841-854 (2005); and Foss et al., Biochemistry 44:15525-15533 (2005)].
The development of fluorescent biosensors to image processes within living cells with high spatial and temporal resolution has transformed what can be accomplished in biological research. [Tsien, Angew. Chem., Int. Ed. 2009, 48:5612]. A commonly used molecule within sensors is the green fluorescent protein (GFP), which must first fold and then undergo an autocatalytic intramolecular chemical reaction to form its chromophore. [Shimomura et al., J. Cell Comp. Physiol. 1962, 59:223; Prasher et al., Gene 1992, 111:229; Chalfie et al., Science 1994, 263:802; Tsien, Annu Rev Biochem 1998, 67:509; Morise et al., Biochemistry 1974, 13:2656; Heim et al., Proc. Natl. Acad. Sci. U.S.A. 1994, 91:12501; and Heim et al., Nature 1995, 373:663]. Although mutants of GFP exhibit emission at a variety of wavelengths [Shaner et al., Nature Biotechnology 2004, 22:1567], there are limits to what can accomplished using mutagenesis to alter their photophysical properties. [Tsien, Angew. Chem., Int. Ed. 2009, 48:5612].
A family of designed compounds are disclosed hereinafter, exemplified by stilbenes that very selectively bind to TTR in human plasma and, when bound, chemoselectively react with the ε-amino group of Lys-15 of TTR, affording an amide bond. These covalent TTR kinetic stabilizers inhibit 10-20 percent more TTR amyloid fibril formation than their non-covalent counterparts, which are exceptional TTR kinetic stabilizers in their own right. The cytotoxicity linked to TTR amyloidogenesis is also inhibited by these covalent TTR kinetic stabilizers.
In addition, certain members of this family of covalent TTR kinetic stabilizers that are themselves non-fluorescent, metal-free compounds bind to and react with TTR creating a fluorophore as a consequence of amide bond conjugation. Owing to the structural plasticity exhibited by the TTR binding site for a wide variety of stilbenes and related molecules, the work underlying the invention disclosed hereinafter indicates that a covalent TTR kinetic stabilizer compound can be extensively modified chemically and the protein modified by mutagenesis to manipulate the excitation and emission wavelengths, the fluorescence lifetime and other photophysical properties of the TTR-conjugate. [Klabunde et al., Nat. Struct. Biol. 2000, 7:312; Petrassi et al., J. Am. Chem. Soc. 2000, 122:2178; Razavi et al., Angew. Chem. Int. Ed. Engl. 2003, 42:2758; Purkey et al., Chem. Biol. 2004, 11:1719; Petrassi et al., et al., J. Am. Chem. Soc. 2005, 127:6662; Johnson et al., Acc. Chem. Res. 2005, 38:911; Johnson et al., J. Med. Chem. 2008, 51:260; Johnson et al., J. Med. Chem. 2008, 51:6348; and Johnson et al., J. Med. Chem. 2009, 52:1115].